Intermediates of sphingolipid (SL) and phospholipid (PL) metabolism serve as second messengers for a number of signaling cascades, including activation of G-protein-coupled receptors such as adrenaline, thrombin, etc., as well as receptor tyrosine kinases by growth factors. These intermediates mediate a number of processes ranging from protein secretion to activation of apoptosis. We have initiated studies to understand different aspects of lipid signaling in Drosophila. Lipid Reservoirs and Signaling. Sphingomyelin (or phosphorylethanolamine ceramide, CPE, in Drosophila) could serve as a reservoir for several lipid messengers such as ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. We have initiated studies to delineate the in vivo role of some of the enzymes of the putative 'Sphingomyelin Cycle'. We have begun by identifying homologous genes in Drosophila. We are using transgenic and mutagenic studies to analyze the importance of such a pathway in Drosophila. We have recently demonstrated that modulation of the sphingolipid biosynthetic pathway, such as targeted expression of ceramidase, rescues degeneration in certain photoreceptor mutants. We have also demonstrated that ceramidase facilitates membrane turnover and rhodopsin endocytosis in Drosophila photoreceptors. Sphingolipids are synthesized vectorially. While the steps that lead up to the generation of ceramide occur in the endoplasmic reticulum (ER), the biosynthesis of sphingomyelin (or CPE in Drosophila) and complex sphingolipids occurs mostly outside of the ER, either in the Golgi complex or in the plasma membrane. This necessitates the active transport of ceramide from ER to the Golgi complex. This transport is mediated by a protein called ceramide transfer protein (CERT). We have now demonstrated that CERT-mediated transfer of ceramide is critical for the biosynthesis of sphingomyelin (or CPE in Drosophila) and complex sphingolipids. Lack of CERT in Drosophila leads to decreased CPE and complex sphingolipids, and plasma membranes with altered physical and physiological properties. These changes render them susceptible to normal loads of reactive oxygen species encountered by the cell. The ensuing oxidative damage to the plasma membrane leads to production of lipid peroxides that will further oxidize the membrane and cellular constituents, leading to a rapid deterioration in the metabolic function of the cell. All these changes manifest as accelerated aging in Drosophila and thus result in a very short life span for these flies. Lipid Distribution and Signaling. PL and SL at the plasma membrane play an important role in stimulus-response coupling, cell differentiation, movement, and exo- and endocytosis. They are asymmetrically distributed in biological membranes, and different proteins catalyzing uni- and bi-directional movements of lipids perpetuate asymmetry. Our current efforts focus on scramblase, a protein proposed to be involved in bi-directional transbilayer movement of phospholipids. We have recently completed two genetic screens and obtained Drosophila flies lacking two of the identified scramblase proteins. We have also generated flies lacking both genes (double mutants). Phenotypic analysis of the double mutants indicates that, surprisingly, scramblases do not have a determining role in the scrambling of phospholipids that accompany apoptosis, phagocytosis and fusion. Instead, scramblases play a regulatory role in regulated exocytosis. This has implications for a wide range of cellular processes involving digestive system and endocrine and exocrine secretions with clinical relevance for a wide range of diseases such as diabetes, behavior disorders and even tumor metastasis. We anticipate that a combination of genetic, molecular, and biochemical approaches in Drosophila will define the important players involved in PL, SL signaling in their normal cellular environment.